Detection method of diamine impurities, minocycline hydrochloride capsule thereof and preparation method of minocycline hydrochloride capsule

Abstract

The invention provides a diamine impurity detection method, a minocycline hydrochloride capsule and a preparation method thereof. The high performance liquid chromatography established by the invention is used for detecting the diamine in the minocycline hydrochloride capsule, the content of the diamine can be accurately determined, and a reliable basis is provided for quality control of the minocycline hydrochloride capsule.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to a method for detecting hydrazine impurities, a minocycline hydrochloride capsule and a preparation method thereof. Background Art

[0002] Minocycline hydrochloride is a semi-synthetic tetracycline broad-spectrum antibiotic developed in the early 1970s. It can reversibly bind to the A site of the 30S subunit of the bacterial ribosome, prevent aminoacyl-tRNA from binding to the receptor site of the mRNA-ribosome complex, inhibit the synthesis of bacterial proteins, and achieve the effect of inhibiting bacterial growth. It is highly effective and long-lasting. It is suitable for infections caused by pathogens such as Staphylococcus, Streptococcus, Pneumococcus, Rickettsia, Chlamydia, Mycoplasma pneumoniae, etc. that are sensitive to minocycline. Among tetracycline antibiotics, minocycline has the strongest antibacterial effect and better antibacterial effect than other antibacterial drugs.

[0003] Genotoxic impurities (or genotoxic impurities, Genotoxic Impurity, GTI) refer to compounds that directly or indirectly damage cell DNA, cause gene mutations or in vivo mutagenesis, and have the potential or tendency to cause cancer.

[0004] Dibenzyl azodicarboxylate is a reagent used in the synthesis of minocycline hydrochloride raw materials. It participates in the reaction of demethyltetracycline to produce 7-HT. 7-HT is a hydrazine compound with a potential genotoxic structure.

[0005] Dibenzyl Hydrazine-1,2-Dicarboxylate, chemical name: 1,2-dibenzyloxycarbonylhydrazine, English name: Dibenzyl Hydrazine-1,2-Dicarboxylate, is the reduction product of dibenzyl azodicarboxylate, also a hydrazine compound, also has a potential genotoxic structure, swallowing, inhalation, skin contact may be harmful to the human body. Therefore, the detection of the content of dibenzyl hydrazine is an important factor in controlling product quality.

[0006] Since hydrazine is irritating, highly flammable, and reactive towards alcohol and water, the development of analytical methods is difficult, and to date there has been no report on the accurate determination of its content. Summary of the invention

[0007] Therefore, the purpose of the present invention is to overcome the defects in the prior art and provide a method for detecting hydrazine impurities, a minocycline hydrochloride capsule and a preparation method thereof. The present invention detects hydrazine in minocycline hydrochloride capsules by high performance liquid chromatography, providing a reliable basis for quality control of minocycline hydrochloride capsules.

[0008] Before describing the present invention, the terms used in this article are defined as follows:

[0009] The term "7-HT" refers to: heptamethine.

[0010] The term "Inertsil Ph-3" refers to: Shimadzu phenyl reversed phase chromatography column, which has phenyl groups directly bonded to silica gel, and the phenyl bonded phase provides π electron interactions between the phenyl groups and the analyte.

[0011] The term "Inersil ODS-3" refers to: Shimadzu C18 reverse phase chromatography column, whose filler is a versatile octadecyl group chemically modified with high purity spherical silica gel, and an ideal end-capping is implemented to provide stronger hydrophobic interactions.

[0012] The term "Waters Atlantis T3" refers to: Waters C18 reversed phase columns that utilize a trifunctional C18 alkyl bonded phase with a bonding density that improves retention of polar compounds and is 100% aqueous mobile phase compatible.

[0013] To achieve the above object, the first aspect of the present invention provides a method for detecting hydrazine impurities in minocycline hydrochloride capsules, wherein the detection method is a high performance liquid chromatography method and preferably comprises the following steps:

[0014] (1) Select the chromatographic column, mobile phase, solvent and UV detector;

[0015] (2) Prepare the minocycline hydrochloride capsule test solution, hydrazine control solution and sensitivity solution respectively;

[0016] (3) According to the chromatographic column, mobile phase, solvent and ultraviolet detector selected in step (1), the sample tray temperature is set, and the minocycline hydrochloride capsule test solution, hydrazine control solution and sensitivity solution prepared in step (2) are respectively injected into the high performance liquid chromatograph for chromatographic analysis to detect the hydrazine impurity in the minocycline hydrochloride capsule.

[0017] According to the detection method of the first aspect of the present invention, in said step (1):

[0018] The chromatographic column is selected from one or more of the following: Inertsil Ph-3, Inersil ODS-3, Waters Atlantis T3, preferably Inertsil Ph-3 or Waters Atlantis T3, most preferably Inertsil Ph-3;

[0019] The mobile phase is phosphate solution and acetonitrile or phosphate solution and methanol, most preferably phosphate solution and acetonitrile; and / or

[0020] The solvent is selected from one or more of the following: an acetonitrile aqueous solution, a methanol aqueous solution, a phosphate solution and a mixed solution of acetonitrile.

[0021] According to the detection method of the first aspect of the present invention, in said step (1):

[0022] The flow rate of the mobile phase is 1.0 mL / min to 2.0 mL / min, preferably 1.0 mL / min to 1.5 mL / min, and most preferably 1.0 mL / min;

[0023] The column temperature of the chromatographic column is 25°C to 35°C, preferably 28°C to 32°C, most preferably 30°C; and / or

[0024] The detection wavelength of the detector is 205 nm to 260 nm, preferably 208 nm to 254 nm, and most preferably 210 nm.

[0025] According to the detection method of the first aspect of the present invention, in said step (1):

[0026] The concentration of the phosphate solution is 0.005-0.03 mol / L, preferably 0.01-0.02 mol / L, and most preferably 0.01 mol / L;

[0027] The pH value of the phosphate solution is 2.5 to 3.8, preferably 2.8 to 3.6, most preferably 3.0; and / or

[0028] When the solvent is a mixed solution of phosphate solution and acetonitrile, the volume ratio of the phosphate solution to acetonitrile is 30-70:30-70, preferably 40-60:40-60, and most preferably 50:50.

[0029] According to the detection method of the first aspect of the present invention, in said step (2):

[0030] The method for preparing the test solution of the minocycline hydrochloride capsule to be tested comprises: taking the minocycline hydrochloride capsule to be tested, weighing it, placing it in a volumetric flask, adding the solvent to dissolve it and diluting it to the scale;

[0031] The preparation method of the hydrazine control solution comprises: taking a hydrazine reference substance, adding the solvent to dissolve, quantifying and diluting; and / or

[0032] The preparation method of the sensitivity solution comprises: taking a hydrazine reference substance, adding the solvent to dissolve it and quantitatively diluting it.

[0033] According to the detection method of the first aspect of the present invention, in said step (2):

[0034] The concentration of minocycline hydrochloride in the tested minocycline hydrochloride capsule test solution is 1.0 mg / mL to 10.0 mg / mL, preferably 2.0 mg / mL to 10.0 mg / mL, and most preferably 10.0 mg / mL;

[0035] The concentration of hydrazine in the hydrazine control solution is 0.05 to 1.0 μg / mL, preferably 0.1 to 0.8 μg / mL, more preferably 0.5 μg / mL; and / or

[0036] The concentration of hydrazine in the sensitivity solution is 0.01-0.2 μg / mL, preferably 0.02-0.15 μg / mL, and most preferably 0.1 μg / mL.

[0037] According to the detection method of the first aspect of the present invention, in said step (3):

[0038] The sample tray temperature is 5 to 28°C, preferably 6 to 25°C, and most preferably 8°C;

[0039] Preferably, when the mobile phase is phosphate solution and acetonitrile or phosphate solution and methanol, the chromatographic analysis comprises gradient elution, and the initial volume ratio of the phosphate solution and acetonitrile or phosphate solution and methanol in the gradient elution is preferably 60-80:20-40, more preferably 55-75:25-35, and most preferably 70:30.

[0040] According to the detection method of the first aspect of the present invention,

[0041] In the chromatogram of the hydrazine control solution, the separation degree between the hydrazine and its adjacent impurity peaks before and after is greater than 3.0, preferably greater than 2.0, most preferably greater than 1.5; and / or

[0042] In the chromatogram of the sensitive solution, the signal-to-noise ratio of the hydrazine peak is greater than 20, preferably greater than 15, and most preferably greater than 10.

[0043] A second aspect of the present invention provides a method for preparing minocycline hydrochloride capsules, the method comprising: detecting hydrazine impurities by high performance liquid chromatography.

[0044] The third aspect of the present invention provides a minocycline hydrochloride capsule, wherein the minocycline hydrochloride capsule is:

[0045] Prepared by detecting hydrazine impurities in minocycline hydrochloride capsules using the method described in the first aspect; or

[0046] The method is described in the second aspect.

[0047] To achieve the above object, the method for detecting genotoxic impurity hydrazine in minocycline hydrochloride capsules by high performance liquid chromatography of the present invention comprises:

[0048] Prepare the test solution and the reference solution, inject them into the high performance liquid chromatograph for chromatographic analysis, and analyze the hydrazine in the test solution according to the chromatogram. Among them, Inersil Ph-3 (4.6mm×250mm, 3μm) chromatographic column was selected, 0.01mol / L phosphate buffer was used as mobile phase A, acetonitrile was used as mobile phase B, 70% acetonitrile was used as solvent, and ultraviolet detector was used as detector.

[0049] In some specific embodiments, the elution procedure is a gradient elution.

[0050] In some specific embodiments, the mobile phase A is adjusted to a pH value of 2.8 to 3.2, preferably 3.0, with phosphoric acid; the column temperature is 28° C. to 32° C., preferably 30° C.; and the detection wavelength is 208 nm to 212 nm, preferably 210 nm.

[0051] In some specific embodiments, the test solution is prepared by the following method: take an appropriate amount of minocycline hydrochloride capsules (approximately equivalent to 500 mg of minocycline), accurately weigh, place in a 50 ml volumetric flask, add a solvent to dissolve and dilute to the scale.

[0052] In some specific embodiments, the reference solution is prepared by the following method: taking an appropriate amount of hydrazine reference, dissolving it in a solvent and quantitatively diluting it to prepare a 0.5 μg / ml solution.

[0053] In some specific embodiments, the method further comprises preparing a sensitivity solution and injecting the solution into a high performance liquid chromatograph for chromatographic analysis.

[0054] In some specific embodiments, the sensitivity solution is prepared by the following method: taking an appropriate amount of a hydrazine reference substance, dissolving it in a solvent, and quantitatively diluting it to prepare a 0.1 μg / ml solution.

[0055] In some specific embodiments, the method further includes system suitability requirements: in the chromatogram of the reference solution, the separation degree of hydrazine and its adjacent impurity peaks before and after should be greater than 1.5; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the hydrazine peak should be greater than 10.

[0056] Compared with the prior art, the method for detecting hydrazine impurities in minocycline hydrochloride capsules of the present invention may have but is not limited to the following beneficial effects:

[0057] The high performance liquid chromatography method established by the invention is used to detect the hydrazine in the minocycline hydrochloride capsules, and the content of the hydrazine can be accurately determined, thus providing a reliable basis for the quality control of the minocycline hydrochloride capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0059] Figure 1 A comparison diagram of the interference test of Example 1 is shown.

[0060] Figure 2 A typical spectrum of the system suitability test of Example 1 is shown (also a chromatogram of the reference substance solution).

[0061] Figure 3 The hydrazine linear relationship graph of Example 1 is shown.

[0062] Figure 4 A typical chromatogram of the spiked test product of Example 1 is shown.

[0063] Figure 5 The chromatogram of the test solution of Example 1 is shown.

[0064] Figure 6 The sensitivity solution chromatogram of Example 1 is shown.

[0065] Figure 7 The test results of the hydrazine impurity in a batch of minocycline hydrochloride capsules in Example 2 are shown.

[0066] Figure 8 The test results of the hydrazine impurity in two batches of minocycline hydrochloride capsules in Example 2 are shown.

[0067] Fig. 9 The test results of hydrazine impurities in three batches of minocycline hydrochloride capsules in Example 2 are shown. DETAILED DESCRIPTION

[0068] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed and specific description and should not be understood as limiting the present invention in any form.

[0069] This section generally describes the materials and test methods used in the experiments of the present invention. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible. It is clear to those skilled in the art that in the context, if not specifically stated, the materials and operating methods used in the present invention are well known in the art.

[0070] The reagents and instruments used in the following examples are as follows:

[0071] Reagents:

[0072] Potassium dihydrogen phosphate was purchased from Guangdong Guanghua Science and Technology Co., Ltd.; phosphoric acid was purchased from Aladdin Reagent (Shanghai) Co., Ltd.; acetonitrile was purchased from Merck KGaA; minocycline hydrochloride capsules were purchased from Haikou Pharmaceutical Factory Co., Ltd.; hydrazine was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0073] instrument:

[0074] High performance liquid chromatography was purchased from Waters Technology (Shanghai) Co., Ltd., model e2695.

[0075] High performance liquid chromatography (HPLC) was purchased from Thermo Fisher Scientific, model U3000.

[0076] pH meter, purchased from Mettler-Toledo, model S220.

[0077] The 1 / 10,000 electronic balance was purchased from Ohaus Instruments (Shanghai) Co., Ltd., model PWN224ZH.

[0078] The 1 / 100,000 electronic balance was purchased from Mettler-Toledo, model XS205DU.

[0079] The one-millionth electronic balance was purchased from Mettler-Toledo, model XP6.

[0080] Example 1

[0081] This example is an exemplary description of the detection method and method verification of the hydrazine impurity in the minocycline hydrochloride capsules of the present invention.

[0082] 1. Chromatographic conditions

[0083] Column: Inertsil Ph-3, 4.6 mm × 250 mm, 3 μm

[0084] Mobile phase: 0.01 mol / L phosphate buffer (1.36 g potassium dihydrogen phosphate, add 1000 ml water to dissolve, and adjust the pH value to 3.0 with phosphoric acid) as mobile phase A; acetonitrile as mobile phase B.

[0085] The elution procedure was gradient elution, and the ratio of mobile phase A to mobile phase B was as follows:

[0086] Table 1 Ratio of mobile phase A and mobile phase B in gradient elution

[0087] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 25 10 90 25.1 70 30 30 70 30

[0088] Solvent: 70% acetonitrile

[0089] Flow rate: 1.0ml / min

[0090] Column temperature: 30°C

[0091] Wavelength: 210nm

[0092] Injection volume: 20 μl

[0093] Sample plate temperature: 8°C

[0094] 2. Determination steps

[0095] (1) Solution preparation

[0096] ① Test solution: Take 1333±0.01 mg of the contents of minocycline hydrochloride capsules (equivalent to 500 mg±0.01 mg of minocycline), accurately weigh, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale.

[0097] ② Reference solution: Take 5±0.001 mg of hydrazine reference substance, dissolve it in solvent and quantitatively dilute it to make a 0.5 μg / ml solution.

[0098] ③Sensitivity solution: Take 5±0.001 mg of hydrazine reference substance, dissolve it in solvent and quantitatively dilute it to make a 0.1μg / ml solution.

[0099] (2) Take 20uL of the reference solution, sensitivity solution and test solution respectively, inject them into the liquid chromatograph and record the chromatogram. The chromatogram of the reference solution is as follows: Figure 2 As shown, the chromatogram of the test solution is Figure 5 As shown, the sensitivity solution chromatogram is as follows Figure 6 shown.

[0100] (3) Calculation method: External standard method. The impurity content is calculated by the following formula (a).

[0101]

[0102] Among them: A 样 A is the peak area of ​​hydrazine in the test solution; 对 is the peak area of ​​the reference solution; N 样 N is the dilution factor of the test solution; 对 W is the dilution multiple of the reference solution; 样 is the sample weight of the test sample, mg; W 对 is the sample weight of the reference substance, mg; M is the average loading amount of the test substance, mg; L is the specification of the test substance; P is the content of the reference substance, %.

[0103] 3. System suitability requirements: In the chromatogram of the reference solution, the separation degree of hydrazine and its adjacent impurity peaks before and after it should be greater than 1.5; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the hydrazine peak should be greater than 10.

[0104] 4. Method Validation

[0105] ① Interference test: Blank solvent, blank excipient and minocycline main component peak have no interference with diamine peak. Interference test comparison chart see Figure 1 .

[0106] ② System suitability test: In the chromatogram of the reference solution, the separation between hydrazine and the adjacent impurity peaks was 41.98, 42.06, 41.06, 41.21, 40.93, and 39.56, respectively; the reference solution was injected 6 times continuously, and the peak area RSD of the hydrazine peak was 0.94%. See Table 2 for details, and the typical spectrum of the system suitability test is shown in Figure 2 .

[0107] ③ Linearity: Take the hydrazine standard substance and prepare solutions containing about 0.1μg, 0.25μg, 0.4μg, 0.5μg, 0.75μg, and 1.0μg of hydrazine per 1ml, respectively. Determine according to the above chromatographic method, and use the concentration (C) as the abscissa and the peak area (A) as the ordinate for linear regression. The regression equation is: y=58261x-202.74; r=1.0000; the y-axis intercept is 0.72 at 100% response value; the linear range is 0.0977μg / ml~0.9773μg / ml. For details, see Figure 3 And Table 3.

[0108] ④ Detection limit and quantification limit: The detection limit and quantification limit of hydrazine are 1.0 ng and 2.0 ng respectively. See Table 4 for details.

[0109] ⑤ Solution stability study: Take the reference solution and the spiked test solution and place them at low temperature of 8°C for 24 hours. The recovery rates of the reference solution and the spiked test solution are within the range of 80% to 120%, indicating that the solution is stable. See Table 5 for details.

[0110] ⑥Precision investigation: Repeatability: The average content of 6 spiked test solutions was 53.6ppm, and the RSD was 1.03% (<10.0%). Intermediate precision: The average content of 6 spiked test solutions was 49.8ppm, and the RSD was 0.78% (<10.0%); the RSD of the content of 12 spiked test solutions was 3.97% (<15.0%). See Table 6 for details.

[0111] ⑦ Accuracy test: The recovery rate of the test solution ranged from 100.95% to 102.72%, with an average recovery rate of 101.8% and an RSD of 0.54%. See Table 7 for details, and the typical chromatogram of the spiked test solution is shown in Figure 4 .

[0112] ⑧ When the wavelength (±2nm), column temperature (±2℃), and phosphate pH value (±0.2) in the chromatographic conditions were slightly changed, the system applicability met the requirements, and the recovery rate of the hydrazine peak area was in the range of 87.25% to 109.63%. See Table 8 for details.

[0113] Table 2 System suitability test results

[0114]

[0115] Table 3 Hydrazine linear relationship test results

[0116]

[0117] Table 4 Detection limit and quantification limit test results

[0118]

[0119] Table 5 Solution stability test results

[0120]

[0121] Table 6 Precision test results

[0122]

[0123] Table 7 hydrazine recovery test results

[0124]

[0125] Table 8 Durability test results

[0126]

[0127] Example 2

[0128] This example is used to illustrate the effect of the method for detecting hydrazine impurities in minocycline hydrochloride capsules of the present invention.

[0129] Three batches of minocycline hydrochloride samples prepared by the applicant were tested for hydrazine according to the method described in Example 1, and the results were all negative. Figure 7 to Figure 9 As shown, this shows that the minocycline hydrochloride drug synthesis process of the applicant's unit has ideal product quality control and does not contain potential genotoxic impurities.

[0130] In Examples 1-2, the detection method of the present invention was repeated on multiple batches of samples, and no possible hydrazine impurities were detected, thereby proving that the minocycline hydrochloride production process of the applicant's unit is safe and stable.

[0131] Example 3

[0132] This example is another exemplary description of the detection method and method validation of hydrazine impurities in minocycline hydrochloride capsules of the present invention.

[0133] 1. Chromatographic conditions

[0134] Column: Waters Atlantis T3, 4.6 mm × 250 mm, 5 μm

[0135] Mobile phase: 0.01 mol / L phosphate solution (take 1.36 g of potassium dihydrogen phosphate, add 1000 mL of water to dissolve, and adjust the pH value to 6.6 with potassium hydroxide) as mobile phase A; methanol is used as mobile phase B.

[0136] The elution procedure was gradient elution, and the ratio of mobile phase A to mobile phase B was as follows:

[0137] Table 9 Ratio of mobile phase A and mobile phase B in gradient elution

[0138] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 25 10 90 25.1 70 30 30 70 30

[0139] Solvent: methanol aqueous solution (methanol: water = 80:20)

[0140] Flow rate: 1.0mL / min

[0141] Column temperature: 30°C

[0142] Wavelength: 254nm

[0143] Injection volume: 20 μl

[0144] Sample plate temperature: 25°C

[0145] Detector: UV detector

[0146] 2. Determination steps

[0147] (1) Solution preparation

[0148] ① Test solution: Take 666±0.01 mg of the contents of minocycline hydrochloride capsules (equivalent to 250 mg±0.01 mg of minocycline), accurately weigh, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale.

[0149] ② Reference solution: Take 2.5±0.001 mg of hydrazine reference substance, dissolve it in solvent and quantitatively dilute it to make a 0.25 μg / mL solution.

[0150] ③Sensitivity solution: Take 2.5±0.001 mg of hydrazine standard substance, dissolve it in solvent and quantitatively dilute it to make a 0.05μg / mL solution.

[0151] (2) Take 20uL of the reference solution, sensitivity solution and test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0152] (3) Calculation method: External standard method. The impurity content is calculated by the following formula (a).

[0153]

[0154] Among them: A 样 A is the peak area of ​​hydrazine in the test solution; 对 is the peak area of ​​the reference solution; N 样 N is the dilution factor of the test solution; 对 W is the dilution multiple of the reference solution; 样 is the sample weight of the test sample, mg; W 对 is the sample weight of the reference substance, mg; M is the average loading amount of the test substance, mg; L is the specification of the test substance; P is the content of the reference substance, %.

[0155] 3. System suitability requirements: In the chromatogram of the reference solution, the separation degree of hydrazine and its adjacent impurity peaks before and after it should be greater than 1.5; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the hydrazine peak should be greater than 10.

[0156] Example 4

[0157] This example is another exemplary description of the detection method and method verification of the hydrazine impurity in the minocycline hydrochloride capsules of the present invention.

[0158] 1. Chromatographic conditions

[0159] Chromatographic column: Inersil ODS-3, 4.6mm×250mm, 5μm

[0160] Mobile phase: 0.01 mol / L phosphate solution (take 1.36 g of potassium dihydrogen phosphate, add 1000 mL of water to dissolve, and adjust the pH value to 3.6 with potassium hydroxide) as mobile phase A; acetonitrile as mobile phase B.

[0161] The elution procedure was gradient elution, and the ratio of mobile phase A to mobile phase B was as follows:

[0162] Table 10 Ratio of mobile phase A and mobile phase B in gradient elution

[0163] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 10 10 90 25 10 90 25.1 70 30 30 70 30

[0164] Solvent: Mobile phase A: Mobile phase B (50:50)

[0165] Flow rate: 1.0mL / min

[0166] Column temperature: 30°C

[0167] Wavelength: 254nm

[0168] Injection volume: 100 μl

[0169] Sample plate temperature: 25°C

[0170] 2. Determination steps

[0171] (1) Solution preparation

[0172] ① Test solution: Take 266±0.01 mg of minocycline hydrochloride capsules (equivalent to 100 mg±0.01 mg of minocycline), accurately weigh, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale.

[0173] ② Reference solution: Take 10.0±0.001 mg of hydrazine reference substance, dissolve it in solvent and quantitatively dilute it to make a 0.1 μg / ml solution.

[0174] ③Sensitivity solution: Take 10.0±0.001 mg of hydrazine standard substance, dissolve it in solvent and quantitatively dilute it to make a 0.02μg / ml solution.

[0175] (2) Take 100uL of the reference solution, sensitivity solution and test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0176] (3) Calculation method: External standard method. The impurity content is calculated by the following formula (a).

[0177]

[0178] Among them: A 样 A is the peak area of ​​hydrazine in the test solution; 对 is the peak area of ​​the reference solution; N 样 N is the dilution factor of the test solution; 对 W is the dilution multiple of the reference solution; 样 is the sample weight of the test sample, mg; W 对 is the sample weight of the reference substance, mg; M is the average loading amount of the test substance, mg; L is the specification of the test substance; P is the content of the reference substance, %.

[0179] 3. System suitability requirements: In the chromatogram of the reference solution, the separation degree of hydrazine and its adjacent impurity peaks before and after it should be greater than 1.5; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the hydrazine peak should be greater than 10.

[0180] According to the detection method of Examples 1-2, the hydrazine reference substances of Examples 3-4 can all be detected, but not detected in the minocycline hydrochloride capsule samples.

[0181] Although the effects of some embodiments are shown above, those skilled in the art should understand that according to the concept of the present invention, the aforementioned other embodiments without specific effects or other technical solutions of the present invention not shown in the embodiments can also achieve the following technical effects stated in the invention content section that are equivalent to the embodiments:

[0182] The high performance liquid chromatography method established by the invention is used to detect the hydrazine in the minocycline hydrochloride capsules, and the content of the hydrazine can be accurately determined, thus providing a reliable basis for the quality control of the minocycline hydrochloride capsules.

[0183] Although the present invention has been described to a certain extent, it is obvious that appropriate changes in various conditions can be made without departing from the spirit and scope of the present invention. It is understood that the present invention is not limited to the embodiments described, but belongs to the scope of the claims, which includes equivalent replacements of each factor described.

Claims

1. A method for detecting hydrazine impurities in minocycline hydrochloride capsules, characterized in that: The detection method is a high performance liquid chromatography method, and preferably comprises the following steps: (1) Select the chromatographic column, mobile phase, solvent and UV detector; (2) Prepare the minocycline hydrochloride capsule test solution, hydrazine control solution and sensitivity solution respectively; (3) According to the chromatographic column, mobile phase, solvent and ultraviolet detector selected in step (1), the sample tray temperature is set, and the minocycline hydrochloride capsule test solution, hydrazine control solution and sensitivity solution prepared in step (2) are respectively injected into the high performance liquid chromatograph for chromatographic analysis to detect the hydrazine impurity in the minocycline hydrochloride capsule.

2. The detection method according to claim 1, characterized in that: In the step (1): The chromatographic column is selected from one or more of the following: Inertsil Ph-3, Inersil ODS-3, Waters Atlantis T3, preferably Inertsil Ph-3 or Waters Atlantis T3, most preferably Inertsil Ph-3; The mobile phase is phosphate solution and acetonitrile or phosphate solution and methanol, most preferably phosphate solution and acetonitrile; and / or The solvent is selected from one or more of the following: an acetonitrile aqueous solution, a methanol aqueous solution, a phosphate solution and a mixed solution of acetonitrile.

3. The detection method according to claim 1 or 2, characterized in that: In the step (1): The flow rate of the mobile phase is 1.0 mL / min to 2.0 mL / min, preferably 1.0 mL / min to 1.5 mL / min, and most preferably 1.0 mL / min; The column temperature of the chromatographic column is 25°C to 35°C, preferably 28°C to 32°C, most preferably 30°C; and / or The detection wavelength of the detector is 205 nm to 260 nm, preferably 208 nm to 254 nm, and most preferably 210 nm.

4. The detection method according to claim 2 or 3, characterized in that: In the step (1): The concentration of the phosphate solution is 0.005-0.03 mol / L, preferably 0.01-0.02 mol / L, and most preferably 0.01 mol / L; The pH value of the phosphate solution is 2.5 to 3.8, preferably 2.8 to 3.6, most preferably 3.0; and / or When the solvent is a mixed solution of phosphate solution and acetonitrile, the volume ratio of the phosphate solution to acetonitrile is 30-70:30-70, preferably 40-60:40-60, and most preferably 50:

50.

5. The detection method according to any one of claims 1 to 5, characterized in that: In the step (2): The method for preparing the test solution of the minocycline hydrochloride capsule to be tested comprises: taking the minocycline hydrochloride capsule to be tested, weighing it, placing it in a volumetric flask, adding the solvent to dissolve it and diluting it to the scale; The preparation method of the hydrazine control solution comprises: taking a hydrazine reference substance, adding the solvent to dissolve, quantifying and diluting; and / or The preparation method of the sensitivity solution comprises: taking a hydrazine reference substance, adding the solvent to dissolve it and quantitatively diluting it.

6. The detection method according to claim 5, characterized in that: In the step (2): The concentration of minocycline hydrochloride in the tested minocycline hydrochloride capsule test solution is 1.0 mg / mL to 10.0 mg / mL, preferably 2.0 mg / mL to 10.0 mg / mL, and most preferably 10.0 mg / mL; The concentration of hydrazine in the hydrazine control solution is 0.05 to 1.0 μg / mL, preferably 0.1 to 0.8 μg / mL, more preferably 0.5 μg / mL; and / or The concentration of hydrazine in the sensitivity solution is 0.01-0.2 μg / mL, preferably 0.02-0.15 μg / mL, and most preferably 0.1 μg / mL.

7. The detection method according to any one of claims 1 to 6, characterized in that: In the step (3): The sample tray temperature is 5 to 28°C, preferably 6 to 25°C, and most preferably 8°C; Preferably, when the mobile phase is phosphate solution and acetonitrile or phosphate solution and methanol, the chromatographic analysis comprises gradient elution, and the initial volume ratio of the phosphate solution and acetonitrile or phosphate solution and methanol in the gradient elution is preferably 60-80:20-40, more preferably 55-75:25-35, and most preferably 70:

30.

8. The detection method according to any one of claims 1 to 7, characterized in that: In the chromatogram of the hydrazine control solution, the separation degree between the hydrazine and its adjacent impurity peaks before and after is greater than 3.0, preferably greater than 2.0, most preferably greater than 1.5; and / or In the chromatogram of the sensitive solution, the signal-to-noise ratio of the hydrazine peak is greater than 20, preferably greater than 15, and most preferably greater than 10.

9. A method for preparing minocycline hydrochloride capsules, characterized in that: The preparation method comprises: detecting hydrazine impurities by high performance liquid chromatography.

10. A minocycline hydrochloride capsule, characterized in that: The minocycline hydrochloride capsules are: The method according to any one of claims 1 to 8 is used to detect hydrazine impurities in minocycline hydrochloride capsules; or Prepared by the preparation method according to claim 9.

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